The Study Of Separation, Purification And Structure Of Neutral Heteropolysaccharides From Smilax Glabra Roxb. And The Mechanism Of Its Immunomodulatory Activity

Objective:1.To select a suitable decolorizing macroporous adsorption resin for Smilar Glabra Roxb. polysaccharides and obtain the optimal decolorization parameters as well.2. To study the extraction, isolation, purification, structure characteristic and physicochemical properties of the new neutral heteropolysaccharides from Smilax Glabra Roxb. in this work.3. To discover the immunomodulatory activity of Smilax Glabra Roxb. neutral heteropolysaccharides of peritoneal macrophages and try to clarity the mechanisms of immunomodulatory activity.Methods:1.with both the decolorization rate and retention rate as the indexs, the decolorization effect of SP by six different resin were studied. And the decolorization conditions, including sample loading, decolorization time, pH, decolorization temperature and decolorization rotation rate, of Smilar Glabra Roxb polysaccharides were optimized using macroporous absorbtion resin by response surface methodology.2. The crude extract of Smilar Glabra Roxb. polysaccharides was obtained by hot water extraction method. To remove the non-sugar impurities from crude polysaccharide by ethanol precipitation, Sevage method and XAD-16 macroporous adsorption resin. And then the hetero-polysaccharide, here named SGRP1, was isolated and purified through DEAE FAST FLOW ion exchange chromatography and Superdex-200 gel column chromatography.3. To investigate the physicochemical properties of SGRP1 by UV spectrum, Coomassie brilliant blue method, phenol-sulfuric acid method, TG analysis and elemental analysis.4. The structure analysis of SGRP1 through determination the relative molecular mass, the monosaccharide composition, periodate oxidation, Smith degradation, methylation, IR spectrum and NMR analysis.5. The effect of SGRP1 on the cell viability of macrophages was measured by MTT method.6. The effect of SGRP1 on the pinocytosis activity of macrophages was determined by neutral red test. And the effect of heteropolysaccharide SGRP1 on the phagocytic activity of macrophages was measured by FITC-labeled E. coli by fluorescence microplate reader, flow cytometry and inverted fluorescence microscope.7.The effect and mechanisms of SGRP1 on the immunomodulatory activity of macrophages were studied by many methods. The concentration of cytokines in the supernatant was detected by ELISA kit and NO detection kit; the gene expression of iNOS, IL-6 and TNF-a were measured by RT-PCR; the expression of JNK and ERK1/2 protein were detected by Western blotting.8. The effect and mechanism of SGRP1 on the secretion of IL-1 β in macrophages was investigated by the methods as follows:the content of IL-1 β in culture supernatant was measured by ELISA kit; Colocalization of NLRP3 and ASC was photographed under a confocal laser scanning microscope (CLSM); the expression of NLRP3 protein was detected by Western blotting.9. The role of Macrophage Scavenger Receptor I (SR), Glucan Receptor (GR), Mannose Receptor (MR), Complement Recetor 3 (CR3) and Toll-Like Receptors (TLR2 and TLR4) on heteropolysaccharide SGRPI-induced macrophage immunomodulation was investigated by ELISA kit to measure the secretion of IL-6 and TNF-α after preincubated wi th these antibody for 2 h and then treated with SGRPI for another 24 h.10. According to the above-mentioned research results, the possible mechanisms of immunomodulatory activity in SGRPI-induced macrophages were summarized and inferred. Results:1. The results showed that the decolorization effects of macroporous resin XAD-16 is the best and its optimal decolorization conditions were described as follows:sample loading was 6 mL, decolorization time was 1.8 h, decolorization temperature was 47.02℃and decolorization rotation rate was 110 r/min. Under these conditions, a decolorization rate of 80.07% and polysaccharide retention rate of 73.52% were obtained. Moreover, the decolorization made Smilar glabra Roxb. polysaccharides with better color.2. Two fraction, SGRP1 and SGRP2, were isolation from the root of Smilar Glabra Roxb. using hot water extraction method.3. SGRP1 is a white velvet like solid, tasteless, soluble in water and insoluble in organic solvents. Its element composition are nitrogen (0.02%), carbon (38.36%) and hydrogen (6.561%). Any nucleic acid and protein substances were not found in UV spectrum of SGRP1. And SGRP1 exhibited good thermostability under extreme heat treatments for its thermal degradation peak is 310.46℃.4. Chemical structure characterization showed that SGRP1, a new heteropolysaccharide, has a molecular weight of 1.26 X 10" Da and consists of mannose, fucose and gluose in molar ratio of 1.00:3.09:39.41. In addition, SGRP1 consists of α-pyranoside and the monosaccharide residues types proven to be (1â†'3)-linked α-L-Fuc, (1â†'3)-linked α-D-Man, (1â†'6)-linked a-D-Glc, (1â†')-linked α-D-Glc.5. SGRP1 has no significant effect on the growth of macrophages (P＞0.05) in the concentration range of 0-500 μg/mL.6. SGRP1 can visibly promote macrophage phagocytic function on neutral red and FITC labeled E. coli, and then can enhance the phagocytic function of mouse murine peritoneal macrophages.7. SGRP1 can remarkably increase the secretion of No, IL-6 and TNF-α of RAW 264.7 cells in a concentration dependent manner. And the molecular mechanism of this activity may be relate to improve the gene expression of iNOS, IL-6 and TNF-a and up-regulation the expression of JNK and ERK 1/2 protein.8. SGRP1 can boost the concentration of inflammatory cytokines IL-1β in culture supernatant and increase the colocalizaiton of NLRP3 with ASC compared with control group and promote the expression of NLRP3 protein. These results revealed that SGRP1 may induce NLRP3 inflammasome formation and activation and thereby lead to secret IL-1β in RAW 264.7 cells.9. After treatment with anti-TLR2/anti-TLR4/anti-CR3/anti-MR/anti-S R/anti-GR, the levels of IL-6 and TNF-α. were all significantly decreased in comparison to the group treated with SGRP1 only (P＜ 0.01). However, the minimum levels of IL-6 and TNF-a were the group treatment of anti-TLR2, this results indicated that TLR2 was the main receptors of SGRP1 on mouse peritoneal macrophage.10. SGRP1 possesses notable immunomodulatory activities and the possible mechanisms were summarized as follows:on the one hand, SGRP1 could be non-specific recognized by many membrane surface receptors and transmit signals into the cell to promote the release pro-inflammatory mediators of NO, TNF-α, IL-6 and IL-1 βvia up-regulating the expression of JNK and ERK1/2 protein and activating the NLRP3 inflammasome in murine peritoneal macrophages, respectively. On the other hand, SGRP1 could significantly enhance the phagocytosis, which play a role in participating in the immune regulation of macrophages. And this may be related to promotion the formation of the lysosomal involving the mannose receptor (MR) to transfer the signal to the RAW 264.7 cells. Conclusions:In the present study, the research results show that a new heteropolysaccharide SGRP1, which has been reported for the first time to possess a complex structure composed of mannose, glucose and fucose, was isolated from Smilax Glabra Roxb. using a series of methods of extract and purification. Meanwhile, SGRP1 is also an ideal immunomodulator, which can provide a scientific basis and a new alternatives for the further development and clinical application of Smilax Glabra Roxb.